Toward Personalised Liquid Biopsies for Urothelial Carcinoma: Characterisation of ddPCR and Urinary cfDNA for the Detection of the TERT 228 G&gt;A/T Mutation

Russo, Ilaria; Ju, Yongwon; Gordon, Naheema S.; Cheng, Kar Keung; James, Nicholas D.; Bryan, Richard T.; Ward, Douglas G.

doi:10.3233/blc-170152

Cited by 45 publications

(40 citation statements)

References 29 publications

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“…Using amplicon sequencing, tumour SMs were detected in 78% of cfDNAs, and cfDNA and cpDNA MAFs were correlated, as previously demonstrated [29]. There was a small (5%) but significant (P < 0.001) increase in average MAF in cfDNA relative to cpDNA.…”

Section: -Gene Panel For Urinary Ubc Diagnosissupporting

confidence: 78%

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

et al. 2019

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Objectives To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell‐pellet (cp)DNA and cell‐free (cf)DNA as part of the development of a non‐invasive clinical assay. Patients and Methods A panel of SMs was validated by targeted deep‐sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture‐based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. Results The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5–98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture‐based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. Conclusions SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23‐gene panel shows promise for the non‐invasive diagnosis and risk stratification of UBC.

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Section: -Gene Panel For Urinary Ubc Diagnosissupporting

confidence: 78%

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

et al. 2019

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“…To extract 1 ng of cfDNA from urine with our protocol costs $2.7 with ZQ, while QC and MM cost $7.6 and $5.0, respectively. ZQ has been widely used for urinary cfDNA isolation in multiple studies of liquid biopsies for urologic malignancies [11,14,33]. In fact, ZQ is designed for both cfDNA and cellular DNA.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Lee

Yoon

et al. 2020

Diagnostics

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Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

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“…There have been several studies on urinary pellet DNA and urine supernatant cfDNA from UBC patients. Various mutations could be detected in urinary pellet DNA, and in urinary cfDNA from UBC. Russo et al reported that 92% of urinary cfDNA analyzed by ddPCR showed the same mutational result as that from the matched tumor tissue analyzed by NGS.…”

Section: Discussionmentioning

confidence: 99%

“…Various mutations could be detected in urinary pellet DNA, and in urinary cfDNA from UBC. Russo et al reported that 92% of urinary cfDNA analyzed by ddPCR showed the same mutational result as that from the matched tumor tissue analyzed by NGS. Togneri et al reported that urinary cfDNA of UBC patients has a higher tumor genomic burden and greater detection potential as a genomic biomarker (90%) than urinary pellet DNA (61%).…”

Section: Discussionmentioning

confidence: 99%

Diagnostic potential of TERT promoter and FGFR3 mutations in urinary cell‐free DNA in upper tract urothelial carcinoma

et al. 2019

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Most upper tract urothelial carcinomas ( UTUC ) are muscle invasive at the time of diagnosis. Current standard methods for the diagnosis of UTUC are invasive. Urine cytology is the only non‐invasive test for detecting UTUC , but its sensitivity is low. A novel non‐invasive assay for UTUC detection would improve patient outcome. This study aimed to investigate the mutation of cell‐free DNA (cf DNA ) in urine supernatant to develop a reliable diagnostic biomarker for UTUC patients. We studied urinary cf DNA from 153 individuals, including 56 patients with localized UTUC , and carried out droplet digital PCR assay for TERT promoter and FGFR 3 hotspot mutations. We could detect mutations of TERT C228T in 22/56 (39.3%), TERT C250T in 4/56 (7.1%), and FGFR 3 S249C in 9/56 (16.1%) patients. FGFR 3 mutation was detected only in ≤ pT 1 tumors (positive predictive value: 100.0%). In combination with cytology results, the sensitivity was 78.6%, and the specificity was 96.0%. Although these data need to be validated in a larger‐scale cohort, mutation analysis of TERT promoter and FGFR 3 in urinary cf DNA has the potential to be a non‐invasive diagnostic marker and reliable factor for tumor staging.

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Toward Personalised Liquid Biopsies for Urothelial Carcinoma: Characterisation of ddPCR and Urinary cfDNA for the Detection of the TERT 228 G>A/T Mutation

Cited by 45 publications

References 29 publications

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23‐gene panel with utility for non‐invasive diagnosis and risk stratification

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnostic potential of TERT promoter and FGFR3 mutations in urinary cell‐free DNA in upper tract urothelial carcinoma

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